Brain acidosis is a common feature in acute neurological diseases particularly in ischemia, and has been assumed to play an important role in the pathology of neuronal injury. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain uncertain, multifactorial and vague. We have substantial preliminary data demonstrating that activation of newly described acid-sensing ion channels (ASICs), members of Degenerin/EnaC superfamily, and subsequent Ca2+ entry through these channels are largely responsible for acidosis-induced, glutamate receptor-independent neuronal injury. In cultured mouse cortical neurons, lowering pH activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are also permeable to Ca2+, and activation of these channels induces increases in the concentration of intracellular Ca2+([Ca2+]i). Activation of ASICs by brief incubation of neurons with acidic solutions induces time-dependent cell injury in the presence of the blockers for both voltage-gated Ca2+ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing the extracellular [Ca2+]. Acid treatment of COS-7 cells that lack functional ASICs does not induce significant cell injury. Similar to the primary cultured neurons, acid treatment induces injury in organotypic brain slices, and the injury of brain slices is inhibited by the blockers of ASICs. Preliminary in vivo studies also demonstrate that intraventricular injection of ASIC1 blocker reduced the infarction volume, and knockout of the ASIC1 gene protects the mouse brain from ischemic injury. Furthermore, our preliminary studies demonstrate that ischemic treatment and metabolic inhibition dramatically potentiate the ASIC currents. This potentiation of ASICs in turn increases acidosis-induced neuronal injury. Our overall objective is to investigate the pathological role of ASICs in the central nervous system and to test the hypothesis that activation of ASICs with subsequent Ca2+ entry is largely responsible for acidosis-mediated, glutamate-independent ischemic brain injury. Specific Aims Aim 1. Ca2+ -permeability of acid-sensing ion channels in CNS neurons Aim 2. Specific subunit configurations are responsible for acidosis-induced neuronal injury Aim 3. Potentiation of ASIC currents by hypoxia/ischemia Aim 4. Neuroprotective role of ASIC blockers or ASIC gene knockout in an in vivo model of brain ischemia and the organotypic brain slices